【摘要】：隨著人們生活習(xí)性和膳食結(jié)構(gòu)的改變,結(jié)直腸癌發(fā)生率已在全球范圍位居第三。葉酸對結(jié)直腸癌在內(nèi)的多種腫瘤有一定防范作用,且能抑制結(jié)直腸癌個體術(shù)后黏膜細(xì)胞增生。FOLR1基因編碼葉酸受體蛋白α(Folate Receptorα,FRα),該受體蛋白與葉酸的親和力及其自身的表達(dá)量對葉酸攝取起決定性作用。FOLR1蛋白能與葉酸特異性地結(jié)合并將其轉(zhuǎn)運至細(xì)胞內(nèi),進(jìn)入細(xì)胞的葉酸通過一碳單位代謝參與DNA的合成、損傷修復(fù)和甲基化。當(dāng)葉酸缺乏和代謝異常時,通常引起DNA結(jié)構(gòu)和基因表達(dá)異常等基因組穩(wěn)定性下降事件,從而提高神經(jīng)系統(tǒng)發(fā)育異常、多種退行性疾病及腫瘤發(fā)生風(fēng)險。為探討不同氧化態(tài)葉酸缺乏對不同狀態(tài)結(jié)腸細(xì)胞FOLR1表達(dá)和細(xì)胞基因組穩(wěn)定性的影響與差異,本研究從葉酸對人正常/結(jié)腸癌細(xì)胞FOLR1轉(zhuǎn)錄與翻譯的影響及差異、葉酸對二受試細(xì)胞染色體不穩(wěn)定性(Chromosome Instability,CIN)的影響二個層面進(jìn)行分析,以RPMI-1640培養(yǎng)基為對照(葉酸2266nmol/L),選取還原態(tài)的5-methyltetrahydrofolate(5-MeTHF)和氧化態(tài)的Folic Acid(FA)缺乏濃度(22.66 nmol/L)、維持基因組結(jié)構(gòu)穩(wěn)定的充足濃度(226.6 nmol/L)干預(yù)培養(yǎng)人結(jié)腸腺癌細(xì)胞COLO205、人正常結(jié)腸上皮細(xì)胞CCD-841-CoN(CCD)28天,取培養(yǎng)第14天和第28天細(xì)胞提取總RNA和總蛋白,用RT-qPCR及2-ΔΔCT法分析受試細(xì)胞二個時段FOLR1基因轉(zhuǎn)錄表達(dá)情況;用Western Blotting檢測二個時段干預(yù)后FOLR1蛋白表達(dá)情況;在干預(yù)培養(yǎng)的第9、18、27天,用胞質(zhì)分裂阻斷微核試驗(Cytokinesis-Block Micronucleus assay,CBMN)檢測二受試細(xì)胞株的CIN水平。研究結(jié)果顯示:(1)在自然狀態(tài)下,CCD細(xì)胞的FOLR1蛋白相對表達(dá)顯著低于COLO205細(xì)胞(P0.001)。(2)二種狀態(tài)的葉酸干預(yù)14和28天,COLO205細(xì)胞FOLR1轉(zhuǎn)錄和蛋白表達(dá)變化趨勢一致;葉酸濃度降低,FOLR1轉(zhuǎn)錄(P0.001~0.01)和蛋白(P0.01~0.05)表達(dá)顯著升高;且在葉酸缺乏(22.66 nmol/L)時,CIN率顯著高于對照(2266nmol/L,P0.01)。在CCD細(xì)胞中,葉酸缺乏(22.66 nmol/L)時,14天干預(yù)時FOLR1轉(zhuǎn)錄(P0.001)和蛋白(P0.01~0.05)較對照組顯著升高;而干預(yù)28天時,葉酸缺乏引起CCD細(xì)胞的FOLR1轉(zhuǎn)錄(P0.001)和蛋白(P0.01~0.05)表達(dá)顯著低于對照,同時,葉酸缺乏引起CCD細(xì)胞的CIN率顯著高于對照組(P0.01~0.05)。(3)不同氧化態(tài)葉酸干預(yù)對COLO205和CCD細(xì)胞FOLR1轉(zhuǎn)錄影響程度存在差異,在葉酸缺乏濃度(22.66 nmol/L)下,5-MeTHF對二種受試細(xì)胞FOLR1轉(zhuǎn)錄表達(dá)上調(diào)作用顯著強于FA組(P0.01)。結(jié)果提示,葉酸缺乏在正常細(xì)胞和結(jié)腸腺癌細(xì)胞中均引起FOLR1 mRNA及蛋白表達(dá)上調(diào),CIN升高;但在正常細(xì)胞中,葉酸持續(xù)缺乏導(dǎo)致后期出現(xiàn)FOLR1mRNA和蛋白表達(dá)下降,推測葉酸缺乏誘導(dǎo)染色體損傷并促使細(xì)胞生長停滯或凋亡,基因表達(dá)下滑;而腫瘤細(xì)胞自身具有永生化及FOLR1高表達(dá)的特性,其響應(yīng)葉酸缺乏及其細(xì)胞生長阻滯效應(yīng)的能力低于正常細(xì)胞。研究還提示,葉酸缺乏導(dǎo)致FOLR1表達(dá)上升,5-MeTHF對基因轉(zhuǎn)錄上調(diào)作用強于FA。
[Abstract]:With the change of people's living habits and dietary patterns, the incidence of colorectal cancer has become the third in the world. Folic acid plays an important role in the prevention of colorectal cancer and inhibits the proliferation of mucosal cells in colorectal cancer patients after operation. The folate receptor protein 偽 (Folate Receptor 偽 (FR 偽) is encoded by the FOLR1 gene, which encodes folate receptor protein 偽-folate receptor 偽 (FR 偽). The affinity of the receptor protein with folic acid and its own expression level play a decisive role in folic acid uptake. FOLR1 protein can specifically bind to folic acid and transport it to cells. Folic acid entering the cells is involved in the synthesis of DNA through the metabolism of one carbon unit. Damage repair and methylation. When folic acid is deficient and abnormal metabolism, it usually leads to genomic stability decline events such as abnormal structure and gene expression of DNA, so as to improve the risk of neurodevelopmental abnormalities, various degenerative diseases and tumorigenesis. In order to investigate the effects and differences of different oxidized folic acid deficiency on the expression of FOLR1 and the stability of cell genome in human normal / colon cancer cells, the effects of folic acid on the transcription and translation of FOLR1 in human normal / colon cancer cells were studied in this study. The effects of folic acid on chromosome instability (Chromosome Instability,CIN) of two tested cells were analyzed on two levels. The RPMI-1640 medium was used as control (folic acid 2266nmol/L). The reduced 5-methyltetrahydrofolate (5-MeTHF) and oxidized Folic Acid (FA) deficiency concentration (22.66 nmol/L) were selected to maintain the stability of genomic structure (226.6 nmol/L) in human colon adenocarcinoma cell line COLO205,. Human normal colon epithelial cells were cultured for 28 days. Total RNA and total protein were extracted from human normal colon epithelial cells on day 14 and day 28. The expression of FOLR1 gene was analyzed by RT-qPCR and 2-螖 CT methods. The expression of FOLR1 protein was detected by Western Blotting, and the CIN level of the two tested cell lines was detected by cytoplasmic mitosis blocking micronucleus test (Cytokinesis-Block Micronucleus assay,CBMN) on the 9th, 18th and 27th day after intervention. The results showed that: (1) under natural condition, the relative expression of FOLR1 protein in CCD cells was significantly lower than that in COLO205 cells (P0.001). (2), and the changes of FOLR1 transcription and protein expression in COLO205 cells were consistent at 14 and 28 days after treatment with folic acid. When folic acid concentration decreased, the expression of FOLR1 transcription (P0.001) and protein (P0.01) increased significantly, and the rate of CIN in folic acid deficiency (22.66 nmol/L) was significantly higher than that in control (2266 nmol / L, P0.01). In CCD cells, FOLR1 transcription (P0.001) and protein (P0.01) at 14 days after folic acid deficiency (22.66 nmol/L) were significantly higher than those of the control group. At 28 days after intervention, folic acid deficiency induced FOLR1 transcription (P0.001) and protein (P0.01) expression in CCD cells significantly lower than that in the control group. The CIN rate of CCD cells induced by folic acid deficiency was significantly higher than that of control group (P0.01 / 0.05). (3). There was a significant difference in the effect of different oxidized folic acid intervention on FOLR1 transcription in COLO205 and CCD cells. At folic acid deficiency concentration (22.66 nmol/L), The upregulated effect of 5-MeTHF on the expression of FOLR1 in the two tested cells was significantly stronger than that in the FA group (P0.01). The results suggested that folic acid deficiency induced the up-regulation of FOLR1 mRNA and protein expression and the increase of CIN in both normal cells and colon adenocarcinoma cells. However, in normal cells, continuous deficiency of folic acid resulted in decreased expression of FOLR1mRNA and protein at the later stage, suggesting that folic acid deficiency induced chromosome damage and cell growth arrest or apoptosis, and decreased gene expression. Tumor cells had immortalization and high expression of FOLR1, and their ability to respond to folic acid deficiency and cell growth arrest was lower than that of normal cells. It is also suggested that folic acid deficiency leads to the increase of FOLR1 expression, and the up-regulation of gene transcription by 5-MeTHF is stronger than that of FA..
【學(xué)位授予單位】：云南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.3

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